Bacillus licheniformis s1brs33 with functions of promoting angelica growth and ligustilide accumulation and application thereof

Abstract

The application discloses a bacillus licheniformis S1BRS33 with functions of promoting growth of angelica sinensis and accumulation of ligustilide, a preparation method and application of the bacillus licheniformis S1BRS33, and belongs to the technical field of microorganisms. The strain is the bacillus licheniformis S1BRS33 with a preservation number of CGMCC No. 38372, and is isolated from rhizosphere soil of the angelica sinensis. The fermentation broth preparation method of the strain is as follows: the strain is inoculated into TSA liquid culture medium, and is cultured at 28 DEG C and 180 rpm in the dark for 72 h, so that the fermentation broth with a viable bacterial concentration greater than 1.0x10 9 cfu / mL is obtained. Experiments show that 100 times dilution liquid of the strain can significantly promote growth of the angelica sinensis, and the root length, stem length, rhizome diameter and dry weight are increased by 31.33%, 127.47%, 39.17% and 50.86% respectively in a pot experiment; meanwhile, the strain can significantly increase the content of ligustilide, a core medicinal component of the angelica sinensis, and the increase rate is 214.82%. The strain is non-pathogenic to the plant, has high ecological safety, and has simple production process, low cost and high industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Bacillus licheniformis S1BRS33 that promotes the growth of Angelica sinensis and the accumulation of its active ingredient ligustilide, and the application of this strain in promoting the growth and development of Angelica sinensis and increasing the content of its active ingredients. Background Technology

[0002] Angelica sinensis (Oliv.) Diels is a perennial herbaceous plant belonging to the Apiaceae family. Its roots are commonly used medicinally and it is widely cultivated in high-altitude areas such as Gansu, Qinghai, and Yunnan provinces, especially in Zhangxian and Minxian counties of Gansu Province. Angelica sinensis holds an important position in traditional Chinese medicine, possessing various effects such as nourishing blood and promoting blood circulation, regulating menstruation and relieving pain, moistening the intestines and promoting bowel movements, and anti-inflammatory and analgesic properties. It is known as the "holy medicine for gynecology." Its chemical components include volatile oils, amino acids, flavonoids, and polysaccharides. Among these, ligustilide in the volatile oil is the main active ingredient and is listed as one of the key standards for evaluating the quality of Angelica sinensis in the Chinese Pharmacopoeia. As the core phthalide component of Angelica sinensis volatile oil, ligustilide has a wide range of pharmacological activities, including anti-inflammatory, antioxidant, antitumor, neuroprotective, and vascular protective effects. It shows particular potential in the prevention and treatment of cardiovascular and cerebrovascular diseases and has broad prospects for clinical application.

[0003] In recent years, with the implementation of the strategy to promote the modernization of traditional Chinese medicine, the market demand and planting area of ​​Angelica sinensis have been expanding year by year. During cultivation, to meet the increasing medicinal demand, producers often use chemical fertilizers to increase the yield of medicinal plants. However, over-reliance on chemical fertilizers leads to serious soil degradation problems, including soil structure damage, compaction, acidification, salinization, and nutrient loss, resulting in a significant decline in soil fertility, reduced ecosystem stability, and ecological degradation. Furthermore, the overuse of chemical pesticides has increased the resistance of pests and diseases, leading to an imbalance in the soil micro-ecosystem and a reduction in microbial diversity, further exacerbating the frequency and severity of pest and disease occurrences. These problems have become important factors affecting the yield and quality of Angelica sinensis, severely restricting its growth and development and the accumulation of its medicinal components. Therefore, developing new, green, and efficient microbial functional agents is of great significance for promoting the sustainable development of medicinal plant production.

[0004] Studies have shown that medicinal plants have formed a mutually beneficial relationship with microorganisms through long-term co-evolution. Microorganisms promote plant growth and development through physiological and metabolic activities such as nitrogen fixation, potassium solubilization, phosphorus solubilization, iron carrier production, and IAA synthesis. Furthermore, microorganisms can enhance plant stress resistance. For example, Bacillus and Pseudomonas species can activate the plant's immune system, alleviating drought, salinity, and heavy metal stress. These microorganisms can also secrete antibiotics and lysozymes, helping plants resist pathogen invasion. In addition, microorganisms can act as "exciter" signals or precursors, influencing the synthesis of medicinal components by regulating secondary metabolic pathways in plants through improvements to the microenvironment. Compared with chemical fertilizers and pesticides, functional microbial agents not only promote plant growth and increase the content of medicinal components but also enhance soil fertility, effectively restore the balance of the soil micro-ecosystem, and reduce environmental pollution.

[0005] Based on this, we screened specific functional strains from the rhizosphere soil of Angelica sinensis that can efficiently promote the growth of Angelica sinensis and significantly increase the accumulation of its core medicinal component, ligustilide, and developed them into microbial agents. This not only provides a reliable means for high-yield and high-efficiency Angelica sinensis, but also provides important support for the sustainable and green development of the Chinese medicinal materials industry, with significant ecological and economic benefits. Summary of the Invention

[0006] To address the existing problems in Angelica sinensis cultivation, such as soil degradation, microecological imbalance, and insufficient accumulation of medicinal components due to over-reliance on chemical fertilizers and pesticides, this invention provides a strain of Bacillus licheniformis S1BRS33 that can significantly promote Angelica sinensis growth and increase the content of its core medicinal component, ligustilide. The invention also provides a method for preparing this strain and its applications. This strain and its fermentation broth can effectively replace chemical fertilizers, providing sustainable technical support for the green and efficient cultivation of Angelica sinensis.

[0007] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0008] The strain described in this invention is Bacillus licheniformis S1BRS33, which was deposited on April 23, 2026, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen Road, Chaoyang District, Beijing, with accession number CGMCCNo. 38372. This strain was isolated from the rhizosphere soil of Angelica sinensis plants in a sample plot in Zhang County, Dingxi City, Gansu Province.

[0009] This invention provides a method for preparing the fermentation broth of Bacillus licheniformis S1BRS33, specifically including the following steps: Inoculating the activated primary strain into TSA liquid culture medium, and incubating it on a shaker set at 28°C and 180 rpm under light-protected conditions for 72 hours, until the viable cell concentration in the liquid fermentation broth reaches greater than 1.0 × 10⁻⁶. 9 The fermentation broth of the strain was obtained by measuring cfu / mL. The formula of the TSA liquid culture medium was: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g sodium chloride, 1000 mL distilled water, and pH 7.0-7.2.

[0010] To further optimize the application effect, the above fermentation broth can be diluted with sterile water to prepare a 100-fold dilution.

[0011] This invention also provides the application of the aforementioned Bacillus licheniformis S1BRS33 and its fermentation broth in the preparation of microbial agents that promote the growth of Angelica sinensis, and in the preparation of microbial agents that increase the content of ligustilide, a medicinal component of Angelica sinensis. Experiments have confirmed that the Bacillus licheniformis S1BRS33 strain provided by this invention has good ecological adaptability, can rapidly colonize the rhizosphere soil and roots of Angelica sinensis, and when applied via root irrigation, can significantly promote the growth and development of Angelica sinensis, while simultaneously and substantially increasing the accumulation of its medicinal component, ligustilide.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects:

[0013] 1. The strain described in this invention can significantly promote the growth and development of Angelica sinensis. In a six-well plate experiment, after treating Angelica sinensis seedlings with 100 times the fermentation broth of this strain, compared with the sterile water control group, the root length promotion rate of Angelica sinensis seedlings reached 187.88%, the stem length promotion rate reached 66.30%, and the dry weight promotion rate reached 150.00%. In a long-term pot experiment, the root length, stem length, rhizome diameter, and dry weight of Angelica sinensis in the S1BRS33 treatment group were significantly increased by 31.33%, 127.47%, 39.17%, and 50.86% respectively compared with the control group, demonstrating excellent growth-promoting effects.

[0014] 2. This invention not only promotes plant growth but also significantly activates the secondary metabolic pathways of Angelica sinensis. High-performance liquid chromatography (HPLC) results from pot experiments showed that treatment with Bacillus licheniformis S1BRS33 fermentation broth significantly increased the content of the core medicinal component ligustilide in Angelica sinensis plants by 214.82% compared to the control group, greatly enhancing the medicinal quality and economic value of Angelica sinensis.

[0015] 3. The strain described in this invention was isolated from the native rhizosphere soil of Angelica sinensis, and it can adapt well to the specific growth environment of Angelica sinensis. It is non-pathogenic to the plant and has no adverse effects on the environment, animals, plants, or human health, demonstrating significant ecological safety. Furthermore, the production process of this strain is simple, the culture medium components are widely available and inexpensive, and the production process is pollution-free, making it valuable for industrial production and large-scale application. Attached Figure Description

[0016] Figure 1 Phylogenetic trees of strain S1BRS33 and related reference strains were constructed using the neighbor-joining method based on the 16S rRNA gene sequence.

[0017] Figure 2 This is a statistical graph showing the effects of Bacillus licheniformis S1BRS33 fermentation broth on the root length, stem length, and dry weight of Angelica sinensis seedlings in a six-well plate experiment.

[0018] Figure 3 This is a visual comparison of the effects of Bacillus licheniformis S1BRS33 fermentation broth on the growth of Angelica sinensis seedlings in a six-well plate experiment.

[0019] Figure 4 This is a visual comparison of the effects of Bacillus licheniformis S1BRS33 fermentation broth on the growth of Angelica sinensis plants in a pot experiment.

[0020] Figure 5 This is the chemical structural formula of ligustilide, a medicinal component of Angelica sinensis.

[0021] Figure 6 Standard curve for determining the content of ligustilide in Angelica sinensis by high performance liquid chromatography.

[0022] Figure 7 The chromatogram for determining the content of ligustilide, a pharmacodynamic component in Angelica sinensis samples by high performance liquid chromatography (left side is the control group TSA, right side is the S1BRS33 treatment group).

[0023] Figure 8 Box plot showing the effect of Bacillus licheniformis S1BRS33 fermentation broth on the accumulation of ligustilide, a medicinal component of Angelica sinensis. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to specific embodiments. However, the embodiments are merely examples and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following specific embodiments are conventional operating methods in the relevant technical field.

[0025] Example 1: Isolation and purification of functional strains

[0026] In September 2022, healthy Angelica rhizosphere soil samples were collected from Kanzhipo Village, Jinzhong Town, Zhang County, Dingxi City, Gansu Province (104°02′04″E, 34°17′26″N). After removing surface debris such as dead branches and leaves, the Angelica roots were gently dug out according to their distribution range (0-30cm). A soil sample from about 1cm of the root system was gently collected using a sterile brush. The collected samples were immediately placed in sterile bags, refrigerated, and brought back to the laboratory.

[0027] The specific method for isolating bacteria S1BRS33 from the rhizosphere soil of Angelica sinensis is as follows: Weigh 1.0 g of fresh soil sample into 10 mL of sterile water, shake to mix, and spread on TSA solid medium according to the 10-fold serial dilution method. Incubate the medium in a constant temperature incubator at 28℃ for 48 hours. Single colonies are picked and purified multiple times using the streak plate method. The composition of the TSA solid medium is as follows: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g sodium chloride, 16.0 g agar, 1000 mL distilled water, pH 7.0-7.2, steam sterilized at 121℃ for 30 min. Mix the purified strain with glycerol (volume ratio 3:1) and store at -80℃.

[0028] Example 2: Molecular biological identification of Bacillus licheniformis S1BRS33

[0029] Genomic DNA was extracted from strain S1BRS33 and amplified by PCR using universal primers 968F and 1401R for the bacterial 16S rRNA gene. The amplified products were detected by agarose gel electrophoresis and then subjected to bidirectional sequencing. The obtained 16S rRNA gene sequence was submitted to the NCBI database for sequence alignment, and multiple sequence alignment was performed using sequences from closely related Bacillus genus reference strains. A phylogenetic tree was constructed using the neighbor-joining method.

[0030] Sequence alignment results showed that strain S1BRS33 had a high degree of 16S rRNA gene sequence similarity to Bacillus licheniformis. Figure 1 As shown, strain S1BRS33 clustered with the reference strain of Bacillus licheniformis. Based on the strain's morphological characteristics, 16S rRNA gene sequence alignment, and phylogenetic analysis, strain S1BRS33 was identified as Bacillus licheniformis. Its 16S rRNA gene sequence has a GenBank accession number of PX736580.

[0031] Example 3: Preparation of fermentation broth of functional strains

[0032] Using a sterile toothpick, a single colony purified in Example 1 was picked up and inoculated into a 500 mL Erlenmeyer flask containing 250 mL of TSA liquid medium. The flask was placed in a shaker at 28°C and 180 rpm and incubated in the dark for 72 hours to obtain the fermentation broth of the strain. At this point, the viable cell concentration in the fermentation broth was greater than 1.0 × 10⁻⁶. 9 cfu / mL.

[0033] Subsequently, the fermentation broth of the strain was diluted to OD using sterile water. 600 The pH value was approximately 0.75-0.85, which was used as the stock solution for the strain. Next, the stock solution was further diluted 100-fold and 200-fold using a 10-fold serial dilution method, and these were used as 100-fold and 200-fold fermentation broths for later use. The TSA liquid culture medium consisted of the following: 15.0 g tryptone, 5.0 g soybean peptone, 5.0 g sodium chloride, 1000 mL distilled water, pH 7.0-7.2, and sterilized by steam at 121℃ for 30 min.

[0034] It should be noted that the fermentation broth used in the six-well plate experiment was the original solution, 100-fold fermentation broth, and 200-fold fermentation broth; the fermentation broth used in the pot experiment was 100-fold fermentation broth.

[0035] Example 4: Effect of Bacillus licheniformis S1BRS33 on the growth of Angelica sinensis in a six-well plate experiment.

[0036] The tested Angelica dahurica variety was “Min Gui No. 1”. Surface-sterilized Angelica dahurica seeds were placed in petri dishes (containing double-layered moistened filter paper) and germinated in the dark at 23℃. Angelica dahurica seedlings with uniform growth were selected for activity experiments.

[0037] A blank control group and treatment groups were added to six-well plates. The blank control group included sterile water (CK1), TSA stock solution (CK2), 100-fold dilution of TSA (CK3), and 200-fold dilution of TSA (CK4). The treatment groups included the original fermentation broth of the strain (T1), 100-fold fermentation broth (T2), and 200-fold fermentation broth (T3). 1 mL of liquid and 6 germinating seedlings were added to each well, with three replicates for each treatment. After sealing the six-well plates with sealing film, they were placed in an artificial climate incubator (temperature: 23℃; photoperiod: 10 h / 14 h) for 7 consecutive days. After the incubation period, the Angelica sinensis seedlings were removed, and their growth indicators—root length, stem length, and dry weight—were measured. The experimental data were statistically analyzed using SigmaPlot 14.0 software.

[0038] The results are as follows Figure 2 , Figure 3As shown in Table 1, compared with the control sterile water (CK1), the fermentation broth of Bacillus licheniformis S1BRS33 diluted 100 times (T2) significantly improved the growth indicators of Angelica sinensis seedlings (P < 0.05), with promotion rates of 187.88%, 66.30% and 150.00% for root length, stem length and dry weight, respectively.

[0039] Table 1. Effects of Bacillus licheniformis S1BRS33 on root length, stem length, and dry weight of Angelica sinensis seedlings.

[0040]

[0041] Example 5: Effects of Bacillus licheniformis S1BRS33 on the growth of Angelica sinensis and the accumulation of its active ingredient ligustilide in a pot experiment.

[0042] 1. Experimental Preparation

[0043] (1) Surface sterilization of Angelica seedlings: Select Angelica seedlings with uniform growth, prepare a 1 g / L carbendazim solution using 50% carbendazim wettable powder, soak the Angelica seedlings for 20 min, and then rinse them 3 times with sterile water.

[0044] (2) Substrate preparation: Black soil from the Angelica sinensis planting base was collected and mixed with vermiculite at a volume ratio of 2:1 to serve as the test soil. The mixed substrate was placed in a high-pressure steam sterilizer for sterilization (121℃, 30 min). Since soil microorganisms are difficult to kill in one go, the sterilization process was repeated 3 times.

[0045] (3) Sterilization of plastic flower pot surface: Select plastic flower pots with a size of 22.4 cm (diameter) × 20.8 cm (height) and use 75% ethanol spray to thoroughly disinfect their surface.

[0046] 2. Experimental treatment and cultivation

[0047] The experiment was conducted in a controlled greenhouse environment. Pretreated Angelica sinensis seedlings were transplanted into sterilized pots, with 5 seedlings per pot, and the temperature was controlled at 25℃ for acclimatization. When the seedlings reached a height of approximately 5 cm, root zone irrigation was initiated. The experiment consisted of two treatment groups, with 3 biological replicates in each group.

[0048] (1) Control group (CK): perfused with TSA blank culture medium diluted 100 times with sterile water;

[0049] (2) Treatment group (S1BRS33): Perfusion with sterile water diluted 100 times, OD 600 Fermentation broth of Bacillus licheniformis S1BRS33 with a value adjusted to 0.75-0.85.

[0050] The angelica was applied via root irrigation, with a single application of 50 mL per plant / pot, repeated every 5 days. After 180 days of continuous cultivation, samples of the angelica plants were collected.

[0051] 3. Detection of ligustilide content

[0052] Weigh 0.5 g of Angelica sinensis root powder, add 50 mL of chromatographic grade methanol, and extract by ultrasonication at 25 ℃ for 30 min; centrifuge at 12,000 r / min for 10 min, and filter through a 0.22 μm filter membrane. A Waters Breeze 2 HPLC system and a Symmetry C18 column were used, with methanol as phase A and 0.2% glacial acetic acid aqueous solution as phase B, a flow rate of 1.0 mL / min, a column temperature of 27 ℃, an injection volume of 20 μL, and a detection wavelength of 310 nm; the gradient was 0 min, A / B = 50 / 50; 9 min, A / B = 65 / 35; and 30 min, A / B = 65 / 35.

[0053] The content of ligustilide in Angelica sinensis was determined by high performance liquid chromatography (HPLC). The regression equation for the established ligustilide standard curve was y = 50972x - 93584, and the coefficient of determination R0 was [value missing]. 2 =0.9991 (see) Figure 6 This achieves precise quantification of the target components (see...). Figure 7 ).

[0054] 4. Experimental Results

[0055] To evaluate the effect of Bacillus licheniformis S1BRS33 on the growth of Angelica sinensis, the experimental results showed that inoculation with Bacillus licheniformis S1BRS33 significantly promoted the growth and development of Angelica sinensis (see Table 2 and...). Figure 4 Compared with the control group, the root length, stem length, rhizome diameter, and dry weight of Angelica sinensis in the treatment group were significantly increased by 31.33%, 127.47%, 39.17%, and 50.86%, respectively.

[0056] Table 2. Effects of Bacillus licheniformis S1BRS33 on growth indicators of Angelica sinensis plants (root length, stem length, rhizome diameter, dry weight)

[0057]

[0058] Note: Lowercase letters a and b represent significant differences between different treatment groups (P < 0.05).

[0059] To clarify the effect of strain S1BRS33 on the accumulation of medicinal components in Angelica sinensis roots, the test results are as follows: Figure 8As shown, after treatment with the bacterial solution described in this invention, the content of medicinal components in Angelica sinensis plants was significantly different from that in the control group (P < 0.05); among them, the content of ligustilide in the treatment group was significantly increased by 214.82% compared with that in the control group.

[0060] These experimental results fully demonstrate that Bacillus licheniformis S1BRS33 not only improves the growth of Angelica sinensis plants, but also significantly enhances the accumulation of its key pharmacological component, ligustilide, showing promising application prospects.

Claims

1. A microbial strain that promotes the growth of Angelica sinensis and the accumulation of its active ingredient ligustilide, characterized in that: The microbial strain is Bacillus licheniformis S1BRS33, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38372.

2. A method for preparing fermentation broth from the microbial strain as described in claim 1, characterized in that, Activated Bacillus licheniformis S1BRS33 was inoculated into TSA liquid medium, the formulation of which was: 15 g tryptone, 5 g soybean peptone, 5 g sodium chloride, and 1000 mL distilled water. Subsequently, the inoculated medium was placed on a shaker set at 34°C and 180 rpm, and cultured under dark conditions for 72 h with shaking to achieve a viable bacterial concentration greater than 1.0 × 10⁻⁶. 9 The standard of cfu / mL is used to obtain the fermentation broth of the strain.

3. The application of Bacillus licheniformis S1BRS33 as described in claim 1, or the fermentation broth prepared by the method described in claim 2, in the preparation of microbial agents that promote the growth of Angelica sinensis.

4. The application of Bacillus licheniformis S1BRS33 as described in claim 1, or the fermentation broth prepared by the method described in claim 2, in the preparation of microbial agents that increase the content of ligustilide, a medicinal component of Angelica sinensis.

5. The application according to claim 3 or 4, characterized in that: The fermentation broth is a diluted solution that has been diluted 100 times with sterile water.

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